Screening and characterization of potential cadmium biosorbent Alcaligenes strain from industrial effluent

160 Journal of Basic Microbiology 2012, 52, 160–166

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Screening and characterization of potential cadmium biosorbent Alcaligenes strain from industrial effluent

Abhijit Kumar1, Swaranjit Singh Cameotra2 and Saurabh Gupta3

1 Department of Microbiology, Dolphin (PG) College of Life Sciences, Chunni Kalan, Punjab, India 2 Institute of Microbial Technology, Chandigarh, India 3 Department of Microbiology, Mata Gujri College, Fatehgarh Sahib, Punjab, India

Cadmium (Cd) is a heavy metal that is easily accumulated in the living organisms in connection with anthropogenic activities which may result in serious health problems. In the present study, five potential cadmium tolerant bacterial strains were isolated from industrial effluent with heavy metal contamination and were screened for biosorption potential with their active growth in different media. After growth in shake flasks containing mineral salt media and tryptone soya broth, cell pellet of AS-5 removed by centrifugation sequestered almost 98% and 69% of cadmium (a.i. 25 mg/l Cd) respectively. Other strains accumulated variable amounts of Cd. 16S rRNA gene sequence of AS-5 and its homology analysis using BLAST reveals its phy-logenetic relationship with family β-proteobacteriaceae and 98% homology with Alcaligenes sp., a facultative anaerobe.

Keywords: Metal sequestration / Biosorption / Bioaccumulation / Cadmium

Received: November 22, 2010; accepted: February 14, 2011

DOI 10.1002/jobm.201000461

Introduction*

Environmental pollution caused by heavy metals has received increasing attention worldwide. Removal of heavy metals from the contaminated soil and water is crucial for environmental protection. Heavy metals removal from contaminated sites is done by precipita-tion, filtration, ion-exchange, oxidation reduction, elec-trochemical recovery, membrane separation and other techniques. Compared with other methods bioremedia-tion, the use of organisms to remove toxic compounds/ heavy metals from contaminated sites is a fast growing and promising technology with several advantages over traditional methods [1]. Cadmium, a heavy metal with no known essential biological functions is one of the most toxic pollutants of the environment [2]. Cadmium becomes a serious pervasive environmental pollutant as it is commonly generated from smelting, mining, elec-troplating and painting industry and is attracting in-creased concern. Ultimately it reaches and accumulates

Correspondence: Saurabh Gupta, Assistant Professor, Department of Microbiology, Mata Gujri College, Fatehgarh Sahib-140407 (Punjab), India E-mail: [email protected] Phone: +919815623983

in tissues of animals and human beings through the food chain [3]. Cadmium is easily taken up by plants, transferred through food chains to cause adverse ef-fects on human health [4]. It is absorbed more effi-ciently by the lungs than by the gastrointestinal tract and accumulates primarily in the liver and kidneys [5]. Kidneys are the primary target of cadmium, and ap-proximately one third of body cadmium is stored in the kidney cortex. Renal cadmium is excreted in urine very slowly. Hence a prolonged half-life of cadmium has been reported in the kidneys [6]. Exposure to higher concentrations leads to renal failure preceding renal tubular dysfunction leading to proteinuria, calciuria, aminoaciduria, glucosuria and tubular necrosis [7]. Inhalation of cadmium results in metal fume fever with chemical pneumonitis, pulmonary edema and death [8]. Micro-organisms have the ability to survive in high metal concentrations by means of their intrinsic prop-erties related to their cell wall structures, production of exo-polysaccharides and lack of transport system [9]. Microbes can develop a specific plasmid and/or chromo-somally determined resistance mechanism that regu-lates the intracellular concentrations of metal ions to

Journal of Basic Microbiology 2012, 52, 160–166 Cadmium biosorption by Alcaligenes strain 161


sub lethal levels. Bacteria employ different strategies for the reduction of cadmium such as metal homeosta-sis, detoxification, an active efflux system which is plasmid encoded and specifically capture and eject undesirable divalent cations through the cell membra-ne [10]. Other strategies include changes in ion perme-ability, adsorption and intra and extracellular complexa-tion and compartmentation [11]. Numerous studies on cadmium biotransformation, in different ecosystems have been reported in recent years and all these studies related to biotransformation of cadmium were carried out under anaerobic conditions. It has been proved that microorganisms are capable of adsorbing heavy metals from aqueous solutions, especially for the metal concentration below 50 mg/l [12]. The metal-binding capacities of several biological materials have been identified to be very high, includ-ing marine algae, fungi and yeasts with a wide range of metal species. There are limited reports on the Cd-sequestration studies using viable and aerobically grow-ing bacterial cells. In the present study, five aerobic strains were isolated with a potential to remediate cadmium contaminated sites. Among these, AS-5 as-signed to Alcaligenes sp. based on the 16S rRNA gene homology has the potential to survive and efficiently accumulate cadmium in natural environments. This is one of the few reports of an Alcaligenes sp. capable of Cd ion sequestration isolated from industrial waste water.

Materials and methods

Enrichment and Isolation of microorganisms Rhizospheric soil and industrial effluent were collected from metal contaminated sites of Punjab. Soil samples were air dried and sieved before isolation of microor-ganisms. Water samples were used as such without any prior treatment. For enrichment and isolation of Cd-tolerant bacteria, soil and water samples were suspend-ed in two different media viz. Tryptone Soya Broth (TSB), composed of (g/l): casein peptone 17.0 g, soymeal peptone 3.0 g, dextrose 2.5 g, sodium chloride 5.0 g, di-potassium hydrogen phosphate 2.5 g and Bushnell Hass Broth (BHB), composed of (g/l): magnesium sulfate 0.2 g, calcium chloride 0.02 g, mono-potassium phosphate 1.0 g, di-ammonium hydrogen phosphate 1.0 g, potas-sium nitrate 1.0 g, ferric chloride 0.05 g supplemented with 0.2% glucose in 100 ml broth after sterilization at 121 °C in autoclave. Cadmium was supplemented after autoclaving using cadmium chloride (cadmium as 1 mg/l a.i.). Flasks were incubated at 37 °C on incubator shaker (120 rpm) until growth appeared. A small aliquot was

then transferred to fresh media containing cadmium for enrichment of cadmium tolerant bacteria with increas-ing concentration of cadmium up to 5 mg/l. After sub-culturing, a loopful of culture was streaked on half strength nutrient agar plates containing cadmium for purification of cultures. Plates were incubated under aerobic conditions at 37 °C for 24 h and colonies with different morphology were streaked on fresh plates in single culture and were preserved for further studies on nutrient agar plates at 4 °C.

Cadmium tolerance Isolated cultures were further inoculated in half strength TSB and BHB containing various concentra-tions of cadmium as active ingredient with cadmium chloride (i.e. 1, 2, 5, 10 and 25 mg/l) to check their tol-erance limits.

Growth profiling All the isolates were exposed to 25 mg/l cadmium as active ingredient contained in CdCl2 form in 100 ml of fresh medium to examine the growth profile of the different organisms with cadmium. 2% inoculum was introduced from overnight grown broth culture into TSB and BHB supplemented with cadmium salt and incubated on an orbital shaker at 120 rpm and 37 °C, along with a control lacking cadmium. Growth was monitored spectrophotometrically by regular measure-ments of optical density, at 600 nm, every 24 h up to 96 h.

Biotransformation studies Mass balance profiling: To check the cadmium bioac-cumulation potential of the isolates, an aliquot of sam-ple was collected from growth associated experiment at the end of the experiment i.e. 96 h for the mass balance of metal in biomass and supernatant. Cadmium uptake by the isolates was determined after separation of the cells by centrifugation (8000 rpm at 4 °C for 10 min) from the spent medium followed by acid digestion and subsequent dilution as described elsewhere [13]. Cell pellet and supernatant containing cadmium were sepa-rately acid digested with premixed concentrated nitric acid (HNO3) and perchloric acid (HClO4) in 3:1 ratio. The samples were digested till white fumes of HNO3 emitted indicating the complete digestion of the sam-ples. After digestion samples were diluted with 0.2% HNO3 and were subjected to ICP-MS analysis. ICP-MS analysis: ICP-MS analysis for estimation of cadmium in different fractions was carried out on Perkin Elmer Sciex Elan DRC-e (Axial field technology). For cadmium, the system was operated under inert

162 A. Kumar et al. Journal of Basic Microbiology 2012, 52, 160–166


conditions maintained with argon gas at a flow rate of 15 l through plasma, 1.2 l and 0.9 l from auxiliary and nebulizer respectively throughout the measurement. Pressure was maintained between 60–70 psi. Plasma formation was carried out at 6000 °C temperature which causes the sample to separate into individual atoms (atomization) followed by ionization with plasma and detection by mass spectrometer.

Biochemical and molecular characterization of selected strain AS-5 was further selected for polyphasic characteri-zation and identification based on the comparative growth profile and bioaccumulation potential of the isolates in TSB and BHB. AS-5 was further characterized in terms of its morphological and biochemical charac-teristics along with molecular identification and phy-logenetic relatedness with other organisms. Biochemi-cal characterization of this strain henceforth referred as Alcaligenes sp. was carried out at the Microbial Type Culture Collection (MTCC-IMTECH), Chandigarh, India (Table 1). Molecular characterization was carried out by isolating genomic DNA using standard protocols [14], amplifying and sequencing the 16S rRNA gene. PCR amplification was carried out using universal primers (27F and 1492R). Initial denaturation was carried at 95 °C for 1 min for 1 cycle followed by 37 cycles (dena-turation 94 °C for 1 min, annealing 55 °C for 2 min, extension 72 °C 3 min) and final extension at 72 °C for 15 min. Amplification of a 1.5 Kb fraction was con-firmed by agarose gel electrophoresis. The PCR product was eluted, purified and sent for partial sequencing at Chromous Biotech, Bangalore. Multiple sequence align-ment was carried out using BLAST [15] followed by classification using (RDP II Classifier tools) and phy-logenetic analysis using MEGA-4 [16].

Statistical analysis Growth and mass balance experiments were carried out in triplicate and results are presented as mean values along with standard error in the respective figures.

Results

Cd-tolerance of isolates Five isolates were found to tolerate 25 mg/l of cadmium (Fig. 1) and were further selected for determination of growth kinetics in presence of cadmium along with biotransformation and metal sequestration studies.

Figure 1. Cadmium tolerance of different isolates.

Cd-confronted growth and metal sequestration studies Growth kinetics of these selected metal-tolerant isolates in cadmium-supplemented broth was monitored by at-tenuance changes over a period of 96 hrs (Fig. 2). The iso-lates showed a more or less similar pattern of growth in Cd2+ containing media. A substantial increase in lag phase of AS-1 was observed in TSB supplemented with cadmium while a marginal effect of cadmium was observed on growth of AS-1 as indicated by comparable growth in positive control as well as cadmium supplemented BHB media (Fig. 2a). An exceptional prolonged lag phase of almost 72 h has been observed with AS-3 in cadmium supplemented TSB as compared to all other isolates (Fig. 2b). An unexpected marginal lag phase was observed in cadmium supplemented BHB media. In all other iso-lates (AS-5, AS-9 and AS-10), a short lag phase was ob-tained with TSB supplemented with cadmium as com-pared to previous two isolates (Fig. 2c–e). Compared growth rate of control and cadmium supplemented TSB was in corroboration with the results of previous isolates. After slow growth, a sudden rise in growth was observed in cadmium supplemented BHB media. In metal sequestration studies, extensive sequestra-tion of metal was observed by strain AS-5, AS-9 and AS-10 as indicated by the concentration of metal in the cell pellet as compared to the supernatant in case of TSB (Fig. 3a). Approximately 69% of metal accumulation has been observed in AS-5 followed by AS-3 and AS-10 re-spectively while a marginal metal sequestration was observed in AS-1 with a transitional accumulation with AS-9. In case of BHB, an opposite trend was noted as all the strains sequester metal efficiently except AS-3 where 61% of metal remained in the supernatant. Ap-proximately 100% biosorption was observed from mass balance studies with AS-1, AS-9 and AS-5 (Fig. 3b).



Figure 2. Comparative growth profile of different isolates with cadmium (25 mg/l) and control in TSB and BHB. (a) AS-1; (b) AS-3; (c) AS-5; (d) AS-9; (e) AS-10.

(a) (b)

(c) (d)

(e)



Figure 3. Cadmium sequestration with different isolates after 96 h. (a): in TSB; (b) in BHB.

Characterization and identification of AS-5 Characterization of isolate AS-5 in terms of morpho-logical, physiological and biochemical characteristics was carried out and is depicted in Table 1. The rod shaped isolate stains Gram-negative, is motile, is a non-spore former and does not produce any pigment. Physiologically, it can tolerate a wide range of pH val-ues (4.0–9.0), a temperature range of 15–42 °C and a high salt concentration (7% w/v). The culture was main-tained and grown under aerobic conditions, is catalase and oxidase positive with no nitrate reduction hence it can be considered to be an obligate aerobe. Analysis of the 16S rRNA gene sequence by multiple sequence alignment (BLAST) indicated 99% homology to Alcali-

genes with 98% alignment coverage over 1.5 kb. The sequence was assigned the GenBank accession number GU726881. Classification of AS-5, as belonging to the genus Alcaligenes, was confirmed using the RDP II Clas-sifier [17]. For phylogenetic profiling, additional se-quences were obtained from GenBank and the Ribo-somal Database Project (RDP II – release 9.58) [18, 19]. Sequences were aligned using Clustal W [20] and phy-logenetic analysis carried out using MEGA 4.0.1 [16] by generating a boot-strap corrected maximum parsimo-nious tree. Sequences were selected based randomly on certain commonly found Alcaligenes and other strains in waste water and industrial effluents (Fig. 4).

Table 1. Morphological, physiological & Biochemical Characteristics of the AS-5.

Characters Observations Characters Observations

Morphological Biochemical characteristics Cell Shape Rods Indole utilization –ve Cell Size 0.3 × 1.2 μm Methyl red –ve Gram Stain Gram-ve Voges Proskeur –ve Motility +ve (peritrichous) Citrate utilization +ve Capsule +ve Nitrate reduction –ve Colony morphology on NA plates

White opaque H2S Production –ve

Pigmentation No pigment Urease –ve Starch hydrolysis –ve Physiological Casein hydrolysis –ve Growth at 4 °C +ve Lipid hydrolysis –ve Growth at 42 °C +ve Gelatin liquefaction –ve Growth at 45 °C –ve Oxidase +ve Growth with 1% NaCl +ve Catalase +ve Growth with 5% NaCl +ve Growth with 7% NaCl +ve Carbohydrate fermentation test Growth with 10% NaCl –ve Sucrose –ve Growth at pH-2 –ve Glucose –ve Growth at pH-5 +ve Lactose –ve Growth at pH-8 +ve Growth at pH-11 –ve

NA – Nutrient agar

(a) (b)



Figure 4. Phyogenetic tree of Alcaligenes sp. with other known and sequenced bacterial strains based on 16S rRNA gene sequence using maximum parsimony analysis.

Discussion

One of our long term research aims is to exploit in-situ organisms for the sequestration of cadmium from in-dustrial effluents, and thus reduce its discharge into natural and large water bodies which otherwise will find their way into biological systems resulting in bio-accumulation and biomagnification. As a part of this study, we isolated five bacterial strains from industrial effluent and are in the process of understanding the mechanism(s) of cadmium transformation by these or-ganisms. The cadmium tolerant bacterial isolates from effluent of metal industry reported in this study may be exploited as an important biological mean in manag-ing the heavy metal pollution through biomass associ-ated sequestration of cadmium resulting in immobili-zation of cadmium. Studies on immobilization of cadmium by facultative bacteria have been reported in diverse variety of species such as Pseudomonas aerugonisa from deep-sea vent sam-ple [21], LAB [22], Rhodotorula sp. Y11 [23] and Bacillus thuringiensis [24]. Although a high tolerance level (up to 5 mM) for Cd-uptake has been published in previous reports [21] in association with complexation with me-dia ingredients. However, in the present study, 98% bioaccumulation was observed in basal media with no chance of complexation. Isolate, AS-5 could grow on

significantly higher concentrations of cadmium than reported in industrial effluent with potential to seques-ter metal by the biomass. Although, the growth in the presence of cadmium was relatively slow, the organism was still observed to accumulate this heavy metal. The isolate AS-5 identified as an Alcaligenes sp. was obtained from effluent of an electroplating industry. Inherited feature of Alcaligenes to form biofilm and potential to uptake and bind heavy metals may facili-tates proper adhesion and efficient immobilization of bacteria to some inert material for in-situ metal seques-tration under pilot scale. The ability of AS-5 to accumu-late cadmium inside the biomass is further evidence of this characteristic of Alcaligenes sp. The data show var-ied growth profiles and affinity of isolates for cadmium salts, with better growth and sequestration with AS-5 as compared to other isolates. Approximately 98% and 69% sequestration of divalent Cd ions was observed in mineral salt media and tryptone soya broth respectively by AS-5 in the cell pellet (membrane bound divalent form or elemental form), on or within the biomass. These results indicate efficient detoxification and trans-formation mechanisms harnessed by these organisms for high concentration of cadmium metal/cation. It also strengthens the notion of complexation of cadmium ions by organic ingredients of TSB resulting in preven-tion of Cd-uptake and a comparative better growth rate.



While speciation of intracellular cadmium was not quantified in the present study, cadmium uptake and assimilation was quite evident. Over 96 hrs, the profile of total cadmium in various fractions viz., cell-free su-pernatant and biomass indicated substantial cadmium up take by the organisms.

Conclusion

A Gram-negative rod shaped bacterial strain has been isolated from the industrial effluent with a potential to tolerate and sequester Cd- metal (as active ingredient) up to 25 mg/l under lab-conditions maintained accord-ing to in-situ environment in terms of nutrient avail-ability. Culture was characterized physiologically, bio-chemically and phylo-genetically and was assigned to Alcaligenes sp. (Accession No. GU726881) after BLAST analysis. The study presents one of the few results among Cd-accumulating bacteria from industrial efflu-ents. Furthermore this indigenous micro flora may be exploited towards the accumulation of heavy metals into the bound form within the biomass for remediat-ing cadmium contamination present in various indus-trial effluents in future.

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Screening and characterization of potential cadmium biosorbent Alcaligenes strain from industrial effluent